DESCRIPTION Abstract Hematopoietic stem cells (HSCs) have the unique capacity to regenerate themselves, as well as to generate all the lineages of the blood. The fine balance between renewal and commitment is effortlessly achieved in vivo. Yet how this balance is regulated is one of the most fundamental unsolved problems in biology. Although in the last four decades we have learned a lot about HSCs, ultimately we have remained incapable of recapitulating what they do in vivo. What's missing is a true view of the signals and the interactions that these cells normally experience in their native microenvironment. Here we propose to study how HSC renewal is controlled by the stem cell niche by imaging HSC interactions with the stromal cell microenvironment, and imaging the signals they receive in vivo, under both homeostatic conditions as well as conditions of regeneration and transformation. To this end, we propose to develop a transgenic system in which hematopoietic stem cells are specifically fluorescently labeled and can be detected in vivo. These cells can then be visualized using live imaging technology to visualize cells within the bone marrow niche in living animals. This would allow a dynamic view of the cell-cell interactions, signaling pathways and cell movements that regulate normal HSC function and transformation. Understanding the in vivo signals and interactions that occur during these processes is key to utilizing these signals for expansion of human hematopoietic stem cells for transplantation. In addition, defining how mechanisms of normal growth and division are properly controlled in stem cells may allow us to find ways to correct their dysregulation in cancer. Delineating the basis of stem cell and cancer cell self-renewal in vivo will in the long term, contribute to novel approaches to regenerative medicine and cancer therapy.